•CRISPR/Cas9-mediated simultaneous targeting of five genes in mES cells

•Generation of Tet1/Tet2 double-mutant mice in one step

•Generation of Tet1/Tet2 double-mutant mice with predefined mutations in one step

Summary

Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty – 8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.

Mice with specific gene modifications are valuable tools for studying development and disease. Traditional gene targeting in mice using embryonic stem (ES) cells, although suitable for generating sophisticated genetic modifications in endogenous genes, is complex and time-consuming. We have recently described CRISPR/Cas-mediated genome engineering for the generation of mice carrying mutations in multiple genes, endogenous reporters, conditional alleles or defined deletions. Here we provide a detailed protocol for embryo manipulation by piezo-driven injection of nucleic acids into the cytoplasm to create gene-modified mice. Beginning with target design, the generation of gene-modified mice can be achieved in as little as 4 weeks. We also describe the application of the CRISPR/Cas technology for the simultaneous editing of multiple genes (five genes or more) after a single transfection of ES cells. The principles described in this protocol have already been applied in rats and primates, and they are applicable to sophisticated genome engineering in species in which ES cells are not available.

Our long-range goals are to understand epigenetic regulation of gene expression in mammalian development and disease. An important question is to understand the different epigenetic conformations that distinguish differentiated cell states and to define strategies to transdifferentiate one differentiated cell type into another. Embryonic stem cells are of major significance because they have the potential to generate any cell type in the body and, therefore, are of great interest for regenerative medicine. A major focus of our work is to understand the molecular mechanisms that allow the reprogramming of somatic cells to an embryonic pluripotent state and to use the potential of patient specific pluripotent cells to study complex human diseases.

CRISPR/Cas9-mediated simultaneous targeting of five genes in mES cells

Generation of Tet1/Tet2 double-mutant mice in one step

Generation of Tet1/Tet2 double-mutant mice with predefined mutations in one step

Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty – 8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.

Genetically modified mice represent a crucial tool for understanding gene function in development and disease. Mutant mice are conventionally generated by insertional mutagenesis (Copeland and Jenkins, 2010; Kool and Berns, 2009) or by gene-targeting methods (Capecchi, 2005). In conventional gene-targeting methods, mutations are introduced through homologous recombination in mouse embryonic stem (ES) cells. Targeted ES cells injected into wild-type (WT) blastocysts can contribute to the germline of chimeric animals, generating mice containing the targeted gene modification (Capecchi, 2005). It is costly and time consuming to produce single-gene knockout mice and even more so to make double-mutant mice. Moreover, in most other mammalian species, no established ES cell lines are available that contribute efficiently to chimeric animals, which greatly limits the genetic studies in many species.

Alternative methods have been developed to accelerate the process of genome modification by directly injecting DNA or mRNA of site-specific nucleases into the one-cell embryo to generate DNA double-strand break (DSB) at a specified locus in various species (Bogdanove and Voytas, 2011; Carroll et al., 2008; Urnov et al., 2010). DSBs induced by these site-specific nucleases can then be repaired by error-prone nonhomologous end joining (NHEJ) resulting in mutant mice and rats carrying deletions or insertions at the cut site (Carbery et al., 2010; Geurts et al., 2009; Sung et al., 2013;Tesson et al., 2011). If a donor plasmid with homology to the ends flanking the DSB is coinjected, high-fidelity homologous recombination can produce animals with targeted integrations (Cui et al., 2011; Meyer et al., 2010). Because these methods require the complex designs of zinc finger nucleases (ZNFs) or Transcription activator-like effector nucleases (TALENs) for each target gene and because the efficiency of targeting may vary substantially, no multiplexed gene targeting in animals has been reported to date. To dissect the functions of gene family members with redundant functions or to analyze epistatic relationships in genetic pathways, mice with two or more mutated genes are required, prompting the development of efficient technology for the generation of animals carrying multiple mutated genes.

Recently, the type II bacterial CRISPR/Cas system has been demonstrated as an efficient gene-targeting technology with the potential for multiplexed genome editing. Bacteria and archaea have evolved an RNA-based adaptive immune system that uses CRISPR (clustered regularly interspaced short palindromic repeat) and Cas (CRISPR-associated) proteins to detect and destroy invading viruses and plasmids (Horvath and Barrangou, 2010; Wiedenheft et al., 2012). Cas proteins, CRISPR RNAs (crRNAs), andtrans-activating crRNA (tracrRNA) form ribonucleoprotein complexes, which target and degrade foreign nucleic acids, guided by crRNAs ( Gasiunas et al., 2012; Jinek et al., 2012). It was shown that the Cas9 endonuclease from Streptococcus pyogenes type II CRISPR/Cas system can be programmed to produce sequence-specific DSB in vitro by providing a synthetic single-guide RNA (sgRNA) consisting of a fusion of crRNA and tracrRNA ( Jinek et al., 2012). More intriguingly, Cas9 and sgRNA are the only components necessary and sufficient for induction of targeted DNA cleavage in cultured human cells ( Cho et al., 2013; Cong et al., 2013; Mali et al., 2013) as well as in zebrafish (Chang et al., 2013; Hwang et al., 2013). A recent report also demonstrated disruption of a GFP transgene in mice using the CRISPR/Cas system ( Shen et al., 2013). The ease of design, construction, and delivery of multiple sgRNAs suggest the possibility of multiplexed genome editing in mammals. Indeed, one study demonstrated that two loci separated by 119 bp could be cleaved simultaneously in cultured human cells at a low efficiency ( Cong et al., 2013). The extent of achievable multiplexed genome editing has yet to be demonstrated in stem cells as well as in animals. Here, we use the CRISPR/Cas system to drive both NHEJ-based gene disruption and homology directed repair (HDR)-based precise gene editing to achieve highly efficient and simultaneous targeting of multiple genes in stem cells and mice.

Simultaneous Targeting up to Five Genes in ES Cells

To test whether the CRISPR/Cas system could produce targeted cleavage in the mouse genome, we transfected plasmids expressing both the mammalian-codon-optimized Cas9 and a sgRNA targeting each gene ( Cong et al., 2013; Mali et al., 2013) into mouse ES cells and determined the targeted cleavage efficiency by the Surveyor assay ( Guschin et al., 2010). All three Cas9-sgRNA transfections produced cleavage at target loci with high efficiency of 36% at Tet1, 48% atTet2, and 36% at Tet3 ( Figure 1B). Because each target locus contains a restriction enzyme recognition site ( Figure 1A), we PCR amplified an ∼500 bp fragment around each target site and digested the PCR products with the respective enzyme. A correctly targeted allele will lose the restriction site, which can be detected by failure to cleave upon enzyme treatment. Using this restriction fragment length polymorphism (RFLP) assay, we screened 48 ES cell clones from each single-targeting experiment. Consistent with the Surveyor analysis, a high percentage of ES cell clones were targeted, with a high probability of having both alleles mutated ( Figure S1A available online). The results summarized in Table 1 demonstrate that between 65% and 81% of the tested ES cell clones carried mutations in the Tet genes with up to 77% having mutations in both alleles.

Figure 1.

Multiplexed Gene Targeting in mouse ES cells

(A) Schematic of the Cas9/sgRNA-targeting sites in Tet1, 2, and 3. The sgRNA-targeting sequence is underlined, and the protospacer-adjacent motif (PAM) sequence is labeled in green. The restriction sites at the target regions are bold and capitalized. Restriction enzymes used for RFLP and Southern blot analysis are shown, and the Southern blot probes are shown as orange boxes.

(D) The sequence of six mutant alleles in triple-targeted ES cell clone 14 and 41. PAM sequence is labeled in red.

(E) Analysis of 5hmC levels in DNA isolated from triple-targeted ES cell clones by dot blot assay using anti-5hmC antibody. A previously characterized DKO clone derived using traditional method is used as a control. See also Figure S1.

(A) RFLP analysis of clones from each single-targeting experiment (1 to 17 are shown).

(B) RFLP analysis of triple-gene-targeted clones (37 to 53 are shown). Tet1 PCR products were digested with SacI, Tet2 PCR products were digested with EcoRV, and Tet3 PCR products were digested with XhoI. WT control is shown in the last lane. Genotyping of clone 51, 52, and 53 are also shown in Figure 1C.

(C) Schematic of the Cas9/sgRNA-targeting sites in Sry and Uty. The sgRNA-targeting sequence is underlined, and the protospacer-adjacent motif (PAM) sequence is labeled in green. The restriction sites at the target regions are bold and capitalized. Restriction enzymes used for RFLP analysis are shown.

(D) RFLP analysis of quintuple-gene-targeted clones (1 to 10 are shown). Sry PCR products were digested with BsaJI, Uty PCR products were digested with AvrII. WT control is shown in the last lane. RFLP analysis of Tet1, 2, 3 loci are not shown.

Plasmids encoding Cas9 and sgRNAs targeting Tet1, Tet2, and Tet3 were transfected separately (single targeting) or in a pool (triple targeting) into ES cells. The number of total alleles mutated in each ES cell clone is listed from 0 to 2 for single-targeting experiment, and 0 to 6 for triple-targeting experiment. The number of clones containing each specific number of mutated alleles is shown in relation to the total number of clones screened in each experiment. See also Table S1.

The high efficiency of single-gene modification prompted us to test the possibility of targeting all three genes simultaneously. For this we cotransfected ES cells with the constructs expressing Cas9 and three sgRNAs targeting Tet1, 2, and 3. Of 96 clones screened using the RFLP assay, 20 clones were identified as having mutations in all six alleles of the three genes ( Figures 1C and S1B and Table 1). To exclude that a PCR bias could give false positive results, we performed Southern blot analysis and confirmed complete agreement with the RFLP results ( Figure 1C). We subcloned and sequenced the PCR products of Tet1-, Tet2-, and Tet3-targeted regions to verify that all of eight tested clones carried biallelic mutations in all three genes with most clones displaying two mutant alleles for each gene with small insertions or deletions (indels) at the target site ( Figure 1D). To test whether these mutant alleles would abolish the function of Tet proteins, we compared the 5hmC level of targeted clones to WT ES cells. Previously, we reported a depletion of 5hmC in Tet1/Tet2 double-knockout ES cells derived using traditional gene-targeting methods ( Dawlaty et al., 2013). As expected from loss of function alleles, we found a significant reduction of 5hmC levels in all clones carrying biallelic mutations in the three genes ( Figure 1E).

To further test the potential of multiplexed gene targeting by CRISPR/Cas system, we designed sgRNAs targeting two Y-linked genes, Sry and Uty ( Figure S1C). Short PCR products encoding sgRNAs targeting all five genes (Tet1, Tet2, Tet3, Sry, and Uty) were pooled and cotransfected with a Cas9 expressing plasmid and the PGK puroR cassette into ES cells. Of 96 clones that were screened using the RFLP assay, 10% carried mutations in all eight alleles of the five genes ( Figure S1D and Table S1), demonstrating the capacity of the CRISP/Cas9 system for highly efficient multiplexed gene targeting.

One-Step Generation of Single-Gene Mutant Mice by Zygote Injection

We tested whether mutant mice could be generated in vivo by direct embryo manipulation. Capped polyadenylated Cas9 mRNA was produced by in vitro transcription and coinjected with sgRNAs. Initially, to determine the optimal concentration of Cas9 mRNA for targeting in vivo, we microinjected varying amounts of Cas9-encoding mRNA with Tet1 targeting sgRNA at constant concentration (20 ng/μl) into pronuclear (PN) stage one-cell mouse embryos and assessed the frequency of altered alleles at the blastocyst stage using the RFLP assay. As expected, higher concentration of Cas9 mRNA led to more efficient gene disruption ( Figure S2A). Nevertheless, even embryos injected with the highest amount of Cas9 mRNA (200 ng/μl) showed normal blastocyst development, suggesting low toxicity.

To investigate whether postnatal mice carrying targeted mutations could be generated, we coinjected sgRNAs targeting Tet1or Tet2 with different concentrations of Cas9 mRNA. Blastocysts derived from the injected embryos were transplanted into foster mothers and newborn pups were obtained. As summarized in Table 2, about 10% of the transferred blastocysts developed to birth independent of the RNA concentrations used for injection suggesting low fetal toxicity of the Cas9 mRNA and sgRNA. RFLP, Southern blot, and sequencing analysis demonstrated that between 50 and 90% of the postnatal mice carried biallelic mutations in either target gene ( Figures 2A, 2B, and 2C and Table 2).

Cas9 mRNA and sgRNAs targeting Tet1, Tet2, or Tet3 were injected into fertilized eggs. The blastocysts derived from injected embryos were transplanted into foster mothers and newborn pups were obtained and genotyped. The number of total alleles mutated in each mouse is listed from 0 to 2. The number of mice containing each specific number of mutated alleles is shown in relation to the total number of mice screened in each experiment. See also Table S2. A Some of the pups were cannibalized.

We also derived blastocysts from zygotes injected with Cas9 mRNA and Tet3 sgRNA. Genotyping of the blastocysts demonstrated that of eight embryos three were homozygous and three were heterozygous Tet3 mutants (two failed to amplify) (Figure S2C). Some blastocysts were implanted into foster mothers and, upon C section, we readily identified multiple mice of smaller size ( Figure S2D), many of which died soon after delivery. Genotyping shown in Figure S2E indicated that all pups with mutations in both Tet3 alleles died neonatally. Only 2 out of 15 mice survived that were either Tet3heterozygous mutants or WT ( Figure S2F). These results are consistent with the lethal neonatal phenotype of Tet3 knockout mice generated using traditional methods ( Gu et al., 2011), although we have not yet established which of the Tet3 mutations produced loss of function rather than hypomorphic alleles.

One-Step Generation of Double-Gene Mutant Mice by Zygote Injection

To test whether Tet1/Tet2 double-mutant mice could be produced from single embryos, we coinjected Tet1 and Tet2 sgRNAs with 20 or 100 ng/μl Cas9 mRNA into zygotes. A total of 28 pups were born from 144 embryos transferred into foster mothers (21% live-birth rate) that had been injected at the zygote stage with high concentrations of RNA (Cas9 mRNA at 100 ng/μl, sgRNAs at 50 ng/μl), consistent with low or no toxicity of the Cas9 mRNA and sgRNAs ( Table 3). RFLP, Southern blot analysis, and sequencing identified 22 mice carrying targeted mutations at all four alleles of the Tet1 and Tet2genes ( Figures 2D and 2E) with the remaining mice carrying mutations in a subset of alleles ( Table 3). Injection of zygotes with low concentration of RNA (Cas9 mRNA at 20 ng/μl, sgRNAs at 20 ng/μl) yielded 19 pups from 75 transferred embryos (25% live-birth rate), which is a higher survival rate than from embryos injected with 100 ng/μl of Cas9 RNA. Nevertheless, more than 50% of the pups were biallelic Tet1/Tet2 double mutants ( Table 3). These results demonstrate that postnatal mice carrying biallelic mutations in two different genes can be generated within one month with high efficiency (Figure 2F).

Cas9 mRNA and sgRNAs targeting Tet1and Tet2 were coinjected into fertilized eggs. The blastocysts derived from the injected embryos were transplanted into foster mothers and newborn pups were obtained and genotyped. The number of total alleles mutated in each mouse is listed from 0 to 4 for Tet1 and Tet2. The number of mice containing each specific number of mutated alleles is shown in relation to the number of total mice screened in each experiment. A Some of the pups were cannibalized.

Although the high live-birth rate and normal development of mutant mice suggest low toxicity of CRISPR/Cas9 system, we sought to determine the off-target effects in vivo. Previous work in vitro, in bacteria, and in cultured human cells suggested that the protospacer-adjacent motif (PAM) sequence NGG and the 8 to 12 base “seed sequence” at the 3′ end of the sgRNA are most important for determining the DNA cleavage specificity (Cong et al., 2013; Jiang et al., 2013; Jinek et al., 2012). Based on this rule, only three and four potential off targets exist in mouse genome for Tet1 and Tet2 sgRNA, respectively ( Table S2 and Experimental Procedures), with each of them perfectly matching the 12 bp seed sequence at the 3′ end and the NGG PAM sequence of the sgRNA (there is no potential off-target site for Tet3 sgRNA using this prediction rule). From seven double-mutant mice produced from injection with high RNA concentration we PCR amplified 400 to 500 bp fragments from all seven potential off-target loci and found no cleavage in the Surveyor assay ( Figure S3), suggesting a high specificity of CRISPR/Cas system.

(A) Three potential off targets of Tet1 sgRNA and four potential off targets of Tet2 sgRNA are shown. The 12 bp perfect matching seed sequence is labeled in blue, and NGG PAM sequence is labeled in red.

(B) Surveyor assay of all seven potential off-target loci in seven double-mutant mice derived with high concentration of Cas9 mRNA (100 ng/μl) injection. WT control is included as the eighth sample. The weak cleavage activity at Ubr1 locus is not due to off-target effect because sequences of these PCR products show no mutations.

The NHEJ-mediated gene mutations described above produced mutant alleles with different and unpredictable insertions and deletions of variable size. We explored the possibility of precise homology directed repair (HDR)-mediated genome editing by coinjecting Cas9 mRNA, sgRNAs, and single-stranded DNA oligos into one-cell embryos. For this we designed an oligo targeting Tet1 so as to change two base pairs of a SacI restriction site and creating instead an EcoRI site and a second oligo targetingTet2 with two base pair changes that would convert an EcoRV site into an EcoRI site (Figure 3A). Blastocysts were derived from zygotes injected with Cas9 mRNA and sgRNAs and oligos targeting Tet1 or Tet2, respectively. DNA was isolated, amplified, and digested with EcoRI to detect oligo-mediated HDR events. Six out of nine Tet1-targeted embryos and 9 out of 15 Tet2-targeted embryos incorporated an EcoRI site at the respective target locus, with several embryos having both alleles modified (Figure S4A). When Cas9 mRNA, sgRNAs, and single-stranded DNA oligos targeting both Tet1 and Tet2 were coinjected into zygotes, out of 14 embryos, four were identified that were targeted with the oligo at the Tet1 locus, seven that were targeted with the oligo at the Tet2 locus and one embryo (2) that had one allele of each gene correctly modified (Figure S4B). All four alleles of embryo 2 were sequenced, confirming that one allele of each gene contained the 2 bp changes directed by the oligo, whereas the other alleles were disrupted by NHEJ-mediated deletion and insertion ( Figure S4C).

Figure 3.

Multiplexed HDR-Mediated Genome Editing In Vivo

(A) Schematic of the oligo-targeting sites at Tet1 and Tet2 loci. The sgRNA-targeting sequence is underlined, and the PAM sequence is labeled in green. Oligo targeting each gene is shown under the target site, with 2 bp changes labeled in red. Restriction enzyme sites used for RFLP analysis are bold and capitalized.

(C) The sequences of both alleles of Tet1 and Tet2 in mouse 5 and 7 show simultaneously HDR-mediated targeting at one allele or two alleles of each gene, and NHEJ-mediated disruption at the other alleles. See also Figure S4.

Blastocysts with double oligo injections were implanted into foster mothers and a total of 10 pups were born from 48 embryos transferred (21% live-birth rate). Upon RFLP analysis using EcoRI, we identified seven mice containing EcoRI sites at the Tet1 locus and eight mice containing EcoRI sites at the Tet2 locus, with six mice containing EcoRI sites at both Tet1 and Tet2 loci ( Figure 3B). We also applied RFLP analysis using SacI and EcoRV to Tet1 and Tet2 loci, respectively, showing that all alleles not targeted by oligos contained disruptions, which is in consistent with the high biallelic mutation rate by Cas9 mRNA and sgRNAs injection. These results were confirmed by sequencing demonstrating mutations in all four alleles of mouse 5 and 7 ( Figure 3C). Our results demonstrate that mice with HDR-mediated precise mutations in multiple genes can be generated in one step by CRISPR/Cas-mediated genome editing.

Discussion

The genetic manipulation of mice is a crucial approach for the study of development and disease. However, the generation of mice with specific mutations is labor intensive and involves gene targeting by homologous recombination in ES cells, the production of chimeric mice, and, after germline transmission of the targeted ES cells, the interbreeding of heterozygous mice to produce the homozygous experimental animals, a process that may take 6 to 12 months or longer (Capecchi, 2005). To produce mice carrying mutations in several genes requires time-consuming intercrossing of single-mutant mice. Similarly, the generation of ES cells carrying homozygous mutations in several genes is usually achieved by sequential targeting, a process that is labor intensive, necessitating multiple consecutive cloning steps to target the genes and to delete the selectable markers.

As summarized in Figure 4, we have established three different approaches for the generation of mice carrying multiple genetic alterations. We demonstrate that CRISPR/Cas-mediated genome editing in ES cells can generate the simultaneous mutations of several genes with high efficiency, a single-step approach allowing the production of cells with mutations in five different genes (Figure 4A). We chose the threeTet genes as targets because the respective mutant phenotypes have been well defined previously ( Dawlaty et al., 2011, 2013; Gu et al., 2011). Cells mutant for Tet1, 2 and 3were depleted of 5hmC as would be expected for loss of function mutations of the genes (Dawlaty et al., 2013). However, we have not as yet established, which of the Cas9-mediated gene mutations produced loss of function rather than hypomorphic alleles.

We also show that mouse embryos can be directly modified by injection of Cas9 mRNA and sgRNA into the fertilized egg resulting in the efficient production of mice carrying biallelic mutations in a given gene. More significantly, coinjection of Cas9 with Tet1 andTet2 sgRNAs into zygotes produced mice that carried mutations in both genes (Figure 4B, upper). We found that up to 95% of newborn mice were biallelic mutant in the targeted gene when single sgRNA was injected and when coinjected with two different sgRNAs, up to 80% carried biallelic mutations in both targeted genes. Thus, mice carrying multiple mutations can be generated within 4 weeks, which is a much shorter time frame than can be achieved by conventional consecutive targeting of genes in ES cells and avoids time-consuming intercrossing of single-mutant mice.

The introduction of DSBs by CRISPR/Cas generates mutant alleles with varying deletions or insertions in contrast to designed precise mutations created by homologous recombination. The introduction of point mutations into human ES cells, cancer cell lines, and mouse by ZNF or TALEN along with DNA oligo has been demonstrated previously (Chen et al., 2011; Soldner et al., 2011; Wefers et al., 2013). We demonstrate that CRISPR/Cas-mediated targeting is useful to generate mutant alleles with predetermined alterations, and coinjection of single-stranded oligos can introduce designed point mutations into two target genes in one step, allowing for multiplexed gene editing in a strictly controlled manner (Figure 4B, lower). It will be of great interest to assess whether this targeting system allows for the production of conditional alleles, or precise insertion of larger DNA fragments such as GFP markers so as to generate conditional knockout and reporter mice for specific genes.

There are several potential limitations of the CRISPR/Cas technology. First, the requirement for a NGG PAM sequence of S. pyogenes Cas9 limits the target space in the mouse genome. It has been shown that the Streptococcus thermophilus LMD-9 Cas9 using different PAM sequence can also induce targeted DNA cleavage in mammalian cells ( Cong et al., 2013). Therefore, exploiting different Cas9 proteins may enable to target most of the mouse genome. Second, although the sgRNAs used here showed high targeting efficiency, much work is needed to elucidate the rules for designing sgRNAs with consistent high targeting efficiency, which is essential for multiplexed genome engineering. Third, although our off-target analysis for the seven most likely off targets of Tet1 and Tet2 sgRNAs failed to detect mutations in these loci, it is possible that other mutations were induced following as yet unidentified rules. A more thorough sequencing analysis for a large number of sgRNAs will provide more information about the potential off-target cleavage of the CRISPR/Cas system and lead to a better prediction of potential off-target sites. Last, oligo-mediated repair allows for precise genome editing, but the other allele is often mutated through NHEJ ( Figures 3B, 3C, andS4C). We have shown that using lower Cas9 mRNA concentration generates more mice with heterozygous mutations. Therefore, it may be possible to optimize the system for more efficient generation of mice with only one oligo -modified allele. In addition, employment of Cas9 nickase will likely avoid this complication because it mainly induces DNA single-strand break, which is typically repaired through HDR ( Cong et al., 2013;Mali et al., 2013).

It is likely that a much larger number of genomic loci than targeted in the present work can be modified simultaneously when pooled sgRNAs are introduced. The methods presented here open up the possibility of systematic genome engineering in mice, facilitating the investigation of entire signaling pathways, of synthetic lethal phenotypes or of genes that have redundant functions. A particularly interesting application is the possibility to produce mice carrying multiple alterations in candidate loci that have been identified in GWAS studies to play a role in the genesis of multigenic diseases. In summary, CRISPR/Cas-mediated genome editing makes possible the generation of ES cells and mice carrying multiple genetic alterations and will facilitate the genetic dissection of development and complex diseases.

Off-target analysis suggests high specificity of the CRISPR/Cas9 system

The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.

Previously, we used the type II bacterial CRISPR/Cas system as an efficient tool to generate mice carrying mutations in multiple genes in one step (Wang et al., 2013). However, this study left a number of issues unresolved. For example, neither the efficiency of using the CRISPR/Cas gene-editing approach for the insertion of DNA constructs into endogenous genes nor its utility to create conditional mutant mice was clarified. Here, we report the one-step generation of mice carrying reporter constructs in three different genes as well as the derivation of conditional mutant mice. In addition, we performed an extensive off-target cleavage analysis and show that off-target mutations are rare in targeted mice and ES cells derived from CRISPR/Cas zygote injection.

Finally, we designed sgRNA targeting the Oct4 3′ UTR, which was coinjected with a published donor vector designed to integrate the 3 kb transgene cassette (IRES-eGFP-loxP-Neo-loxP; Figure 2D) at the 3′ end of the Oct4 gene ( Lengner et al., 2007). Blastocysts were derived from injected zygotes, inspected for GFP expression, and explanted to derive ES cells. About 20% (47/254) of the blastocysts displayed uniform GFP expression in the ICM region. Three of nine derived ES cell lines expressed GFP (Figure 2E), including one showed mosaic expression ( Table S2). Three out of ten live-born mice contained the targeted allele ( Table 1). Correct targeting in mice and ES cell lines was confirmed by Southern blot analysis ( Figure 2F).

Conventionally, transgenic mice are generated by pronuclear instead of cytoplasmic injection of DNA. To optimize the generation of CRISPR/Cas9-targeted embryos, we compared different concentrations of RNA and the Nanog-mCherry or the Oct4-GFP DNA vectors as well as three different delivery modes: (1) simultaneous injection of all constructs into the cytoplasm, (2) simultaneous injection of the RNA and the DNA into the pronucleus, and (3) injection of Cas9/sgRNA into the cytoplasm followed 2 hr later by pronuclear injection of the DNA vector. Table S1 shows that simultaneous injection of all constructs into the cytoplasm at a concentration of 100 ng/μl Cas9 RNA, 50 ng/μl of sgRNA and 200 ng/μl of vector DNA was optimal, resulting in 9% (86/936) to 19% (47/254) of targeted blastocysts. Similarly, the simultaneous injection of 5 ng/μl Cas9 RNA, 2.5 ng/μl of sgRNA, and 10 ng/μl of DNA vector into the pronucleus yielded between 9% (7/75) and 18% (13/72) targeted blastocysts. In contrast, the two-step procedure with Cas9 and sgRNA simultaneous injected into the cytoplasm followed 2 hr later by pronuclear injection of different concentrations of DNA vector yielded no or at most 3% (1/34) positive blastocysts. Thus, our results suggest that simultaneous injection of RNA and DNA into the cytoplasm or nucleus is the most efficient procedure to achieve targeted insertion.

A total of 98 E13.5 embryos and mice were generated from zygotes injected with Cas9 mRNA, sgRNAs, and DNA oligos targeting the L2 and R1 sites. Genomic DNA was digested with both NheI and EcoRI, and analyzed by Southern blot using exon 3 and exon 4 probes (Figures 3A and 3B). The L2 and R1 oligos contained, in addition to the loxP site, different restriction sites (NheI or EcoRI). Thus, single loxP site integration at L2 or R1 will produce either a 3.9 kb or a 2 kb band, respectively, when hybridized with the exon3 probe (Figures 3A and B). We found that about 50% (45/98) of the embryos and mice carried a loxP site at the L2 site and about 25% (25/98) at the R1 site. Importantly, integration of both loxP sites on the same DNA molecule, generating a floxed allele, produces a 700 bp band as detected by exon 3 probe hybridization (Figures 3A and 3B). RFLP analysis, sequencing (Figures S4A and S4B) and Southern blot analysis (Figure 3B) showed that 16 out of the 98 mice tested contained two loxP sites flanking exon 3 on the same allele. Table 2 summarizes the frequency of all alleles and shows that the overall insertion frequency of an L2 or R1 insertion was slightly higher in females (21/38) than in males (28/60) consistent with the higher copy number of the X-linkedMecp2 gene in females. To confirm that the floxed allele was functional, we used genomic DNA for in vitro Cre-mediated recombination. Upon Cre treatment, both the deletion and circular products were detected by PCR in targeted mice, but not in DNA from wild-type mice ( Figure 3C). The PCR products were sequenced and confirmed the precise Cre-loxP-mediated recombination ( Figure S4C).

In this study, we demonstrate that CRISPR/Cas technology can be used for efficient one-step insertions of a short epitope or longer fluorescent tags into precise genomic locations, which will facilitate the generation of mice carrying reporters in endogenous genes. Mice and embryos carrying reporter constructs in the Sox2, the Nanog and the Oct4 gene were derived from zygotes injected with Cas9 mRNA, sgRNAs, and DNA oligos or vectors encoding a tag or a fluorescent marker. Moreover, microinjection of two Mecp2-specific sgRNAs, Cas9 mRNA, and two different oligos encoding loxP sites into fertilized eggs allowed for the one-step generation of conditional mutant mice. In addition, we show that the introduction of two spaced sgRNAs targeting the Mecp2 gene can produce mice carrying defined deletions of about 700 bp. Though all RNA and DNA constructs were injected into the cytoplasm or nucleus of zygotes, the gene modification events could happen at the one-cell stage or later. Indeed, Southern analyses revealed mosaicism in 17% (1/6) to 40% (20/49) of the targeted mice and ES cell lines indicating that the insertion of the transgenes had occurred after the zygote stage ( Table S2).

More…

In summary, CRISPR/Cas-mediated genome editing represents an efficient and simple method of generating sophisticated genetic modifications in mice such as conditional alleles and endogenous reporters in one step. The principles described in this study could be directly adapted to other mammalian species, opening the possibility of sophisticated genome engineering in many species where ES cells are not available.

Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system

Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 (dCas9) protein fused with a transcriptional activation domain and single guide RNAs (sgRNAs) with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore, we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4genes. The most efficient gene activation was achieved by clusters of 3-4 sgRNAs binding to the proximal promoters, suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system.

Gene expression is strictly controlled in many biol-ogical processes, such as development and diseases. Transcription factors regulate gene expression by binding to specific DNA sequences at the enhancer and promoter regions of target genes, and modulate transcription through their effector domains1. Based on the same principle, artificial transcription factors (ATFs) have been generated by fusing various functional domains to a DNA binding domain engineered to bind to the genes of interest, thereby modulating their expression2,3. The capability of regulating endogenous gene expression using ATFs may facilitate the study of the transcriptional network underlying complex biological processes and provide new therapeutic options for diseases. Significant efforts and progress have been made to engineer DNA binding domains with defined specificities. The decipherment of the “code” of DNA binding specificity of zinc finger proteins and transcription activator-like effectors (TALE) has led to the rational design of DNA binding domains to recognize specific nucleotides with certain probability4,5,6,7,8,9,10. However, binding specificity of these ATFs is usually degenerate, can be difficult to predict and the complex and time-consuming design and generation limits their applications. To study the transcriptional network in a systematic manner, regulating multiple endogenous genes is required, prompting the development of efficient technology for simultaneous regulation of multiple endogenous genes.

CRISPR (clustered regularly interspaced short palin-dromic repeat) and Cas (CRISPR-associated) proteins are utilized by bacteria and archea to defend against viral pathogens11,12. Because the binding of Cas protein is guided by the simple base-pair complementarities between the engineered single guide RNA (sgRNA) and a target genomic DNA sequence, Cas9 could be directed to specific genomic locus or multiple loci simultaneously, by providing the engineered sgRNAs13,14,15,16,17,18,19,20. A recent study described the CRISPRi (CRISPR interference) system, in which the nuclease-deficient dCas9 (D10A; H840A) proteins blocked the transcription apparatus when directed to promoters or gene bodies in bacteria21. A subsequent study demonstrated a more efficient gene repression in eukaryotes by dCas9 fused with a transcription repression domain or exogenous transgene activation when fused with an activation domain22. Two most recent studies showed single endogenous gene activation using dCas9-based activators9,10. To what extent multiple endogenous genes could be regulated simultaneously has not been explored. In this study we report the generation of an RNA-programmable CRISPR-on system, which enables the simultaneous activation of multiple endogenous genes with a defined stoichiometry.

…

We show here that the CRISPR-on system can be used for the simultaneous induction of at least three different endogenous genes. More significantly, we demonstrated that the stoichiometry of gene induction of multiple genes can be tuned by adjusting the relative amount of their cognate sgRNAs. Simultaneous activation of multiple endogenous genes with defined stoichiometry opens up novel opportunities for systems biology as it allows for the predictable manipulation of transcriptional networks.

Finally, with the ease of design and synthesis, a library of sgRNAs could be generated. When introduced into a cell line constitutively expressing dCas9 activator, gene activation screens mediated by RNA (RNAa) could be achieved. As the specificity components (sgRNA) can be separately designed and constructed from the effector component (Cas fusion proteins), the same library of sgRNAs could be used with different dCas9 fusions (e.g., VP160 domain for transactivation, KRAB domain for transcriptional repression, chromatin modifier domains for specific histone modification) to exert different functions at particular genomic loci.

Our long-range goals are to understand epigenetic regulation of gene expression in mammalian development and disease. An important question is to understand the different epigenetic conformations that distinguish differentiated cell states and to define strategies to transdifferentiate one differentiated cell type into another. Embryonic stem cells are of major significance because they have the potential to generate any cell type in the body and, therefore, are of great interest for regenerative medicine. A major focus of our work is to understand the molecular mechanisms that allow the reprogramming of somatic cells to an embryonic pluripotent state and to use the potential of patient specific pluripotent cells to study complex human diseases.

Three years ago, no one knew or cared about much about a protein called Cpf1 produced by a bacterial gene. Now, it shows potential for making a fast-developing genome editing technique called CRISPR easier and more accurate. Bioinformaticians identified this protein and its potential connection to CRISPR by scanning the public database of genome sequences. Their colleagues now show that two of 16 versions of this protein tested can delete a gene in a human cell. Cpf1 has other advantages as well-being smaller than one of the popular Cas9 proteins used and depending on a smaller piece of RNA to find its target DNA. But its utility for editing genomes of human and other cells needs further testing.